Skin fibrosis in systemic sclerosis (SSc) is associated with increased expression of genes activated by transforming growth factor-beta (TGF2) and the appearance of myofibroblasts, a cell type induced from fibroblasts by TGF2. The long-term goal of this grant is to understand how TGF2 activity is upregulated in SSc skin. The parent R01 for this competitive revision has been examining pathogenesis and TGF2 activation in Tsk mice, a murine model for SSc skin fibrosis. Work from this grant has shown that developmental regulators, particularly Wnt and BMPs, likely affect skin development perinatally, leading to fascia thickening in Tsk mice, but did not find upregulation of TGF2-responsive genes and no evidence of inflammation in Tsk skin. In other work we have identified biomarkers in SSc skin, showing that increased TGF2- and interferon (IFN)-responsive gene expression in skin correlate highly with the extent of skin disease, indicating a close link between TGF2 activation and inflammation, and suggesting innate immune/toll-like receptor (TLR) activation in SSc skin. Thus, this application investigates a new model of TLR-dependent skin fibrosis developed during the previous funding period that extends studies in Tsk mice to a model that is dependent on TGF2, shows skin inflammation and fibrosis, and is stimulated by TLR ligands. We propose studies to investigate innate immune stimulated dermal fibrosis using this murine model, in which osmotic pumps containing poly(I:C), a TLR3 and RIGI-like receptor agonist, is implanted subcutaneously in mice. In the first aim, proposed experiments will examine the importance of T cells, macrophages and dendritic cells, using rag1 deficient or transgenic mice inducibly deleted of macrophages or dendritic cells. Proposed experiments will also investigate the roles of TH1 and TH2 cytokines: IFN3, IL-4 and IL-13 in poly(I:C)-induced pump dermal fibrosis, using knock-out mice deleted of these cytokines or cytokine receptors. The second aim will investigate the dependence of this model on TLR3 and intracellular TRIF signaling compared to non-TLR mediated signaling through IPS-1. Additional experiments will investigate the mechanisms, increased secretion or activation, and pathways, including NF:B, MAPK and IRF3, utilized by poly(I:C) to stimulate fibroblast TGF2 activity in vitro. Finally, experiments in aim 3 will examine how genetic markers associated with SSc, IRF5 and stat4, influence dermal inflammation and fibrosis using the poly(I:C) pump fibrosis model. Together, these results should significantly extend our understanding of the roles of TH1 and TH2 skewing and associated cytokines in dermal fibrosis, shed light on how different TLR ligands and SSc-associated genes regulate inflammation and associated TGF2 activation in SSc skin, and provide proof-of-principal data for emerging therapeutics targeting TH2 cytokines and TLRs. PUBLIC HEALTH RELEVANCE: Systemic sclerosis (SSc) is a sometimes fatal illness characterized by fibrosis and vascular ischemia affecting skin and internal organs. Dermal fibrosis in SSc frequently causes severe disability, with loss of joint mobility sometimes leading to fixed flexed joint contractures and painful, disfiguring skin disease. Better understanding skin fibrosis will likely also provide further insights into pulmonary fibrosis, a common cause for death of these patients. We have found that the extent of skin disease is associated with increased expression of genes typically regulated by transforming growth factor-beta and type I interferons. In this proposal, we will investigate mechanisms promoting fibrosis in a mouse model we have recently developed for this disease that mimics the alterations in gene expression seen in SSc skin. The results should directly impact the health of these patients by providing guidance for therapeutics currently under development that target the cytokines that appear to be regulating these genes and causing fibrosis.